DMA replication, recombination, and repair are processes fundamental for the transmission of genetic information from one generation of cells to the next. These processes require that duplex DMA is transiently unwound to form a single-stranded intermediate. The unwinding reaction is catalyzed by a class of enzymes called helicases. Helicases are essential for all aspects of nucleic acid metabolism in which ss nucleic acid intermediates are required. Therefore, it is of fundamental importance to understand the molecular mechanism by which these enzymes function in performing their activities. Knowledge of the mechanistic details of the reactions catalyzed by helicases is essential for our understanding of why such processes dysfunction in various diseases; e.g.; cancer and human genetic diseases. Studying different steps on the molecular level will provide the necessary knowledge about how to regulate and control them. This knowledge, in turn, will be very useful in designing efficient therapies for diseases. As a primary replicative helicase in E. Coli cells, the DnaB protein provides an outstanding and paradigm model system to study the molecular mechanism of the replicative helicase. The replicative helicase does not act alone. In the cell, the DnaB helicase is linked to the replication apparatus through the specific replication factor, the DnaC protein. .The DnaB - DnaC complex constitutes a fundamental model of the role of a specific replication factor that connects the helicase to the rest of the replication machine and controls the activities of the enzyme. This research project has three major objectives: The first major objective is to examine the dynamics and energetics of the conformational heterogeneity of the DnaB hexamer and its complex with the DnaC protein. This objective can be achieved by quantitatively examining the thermodynamics and kinetics of conformational transitions and assembly reactions of the DnaB and DnaB-DnaC complex. The second objective is to determine the DnaB - DnaC complex interactions with the ssDNA and the replication fork and the mechanism of the dsDNA unwinding. This objective can be achieved by obtaining detailed kinetics of the individual steps involved in the DNA recognition and unwinding reactions. The third major objective is to determine the dynamics of the formation of DnaB - DnaC complexes and the role of ATP, ADP, and the DNA in this process. To achieve these goals, we will apply steady-state, lifetime fluorescence spectroscopy, the fluorescence energy transfer method, fast kinetic (stopped-flow, rapid quench-flow) methods, dynamic light scattering and analytical ultracentrifugation.